The long-term goal of this project is to understand the molecular basis of bacterial unculturability. ~99% of all bacteria do not grow on synthetic media in vitro, which seriously undermines progress in microbiology, including critical areas related to human health. The established paradigm holds that unculturable organisms are either extremely slow growers, or require some unknown nutrients. In this proposal, we will test an alternative signaling hypothesis of unculturability - most microorganisms evolved to grow in a familiar environment, and will only propagate in response to signals from their surroundings. We have previously developed a method to grow unculturable bacteria by culturing them in a diffusion chamber placed in their natural environment (Kaeberlein, T., Lewis, K., and Epstein, S.S. 2002. Isolating "uncultivable" microorganisms in pure culture using a simulated natural environment. Science 296:1127-1129). Our preliminary findings indicate that growth on synthetic media of isolates from the chamber can be achieved by selection for domesticated variants; or in co-culture with "helper" species. These preliminary observations are consistent with the signaling hypothesis and form the basis of the present proposal. The goal of this project is to discover the genes controlling culturability of a model microorganism MSC33 related to culturable Psychrobacter. We were able to isolate a "domesticated" variant of MSC33 that grows well on a variety of synthetic media. This model will enable us to identify the genetic differences between the parent unculturable and the culturable derivative organisms. This work will be facilitated by a shuttle vector we developed based on an endogenous plasmid from MSC33. Finding the first culturability genes is the essential step toward elucidating the signal transduction pathway controlling growth which we will undertake in a subsequent RO1 project. The reasons preventing the majority of microorganisms from growing in the lab are unknown, and microbiology as a discipline has been restricted to the study of 1% of bacterial species. This project will advance our understanding of uncultivable bacteria, and will help develop tools to grow them. Unculturable bacteria are an enormous untapped source of potentially useful pharmaceutical compounds. Unculturable bacteria also make up most of the human oral and intestinal microflora. This project will advance our understanding of uncultivable bacteria, and will help develop tools to grow them. Unculturable bacteria are an enormous untapped source of potentially useful pharmaceutical compounds. Unculturable bacteria also make up most of the human oral and intestinal microflora. [unreadable] [unreadable] [unreadable]